The serine/threonine protein kinase p381 MAPK is an established therapeutic target for a number of peripheral inflammatory diseases where increased proinflammatory cytokine production contributes to pathology. In contrast, much less is known about the in vivo role of p381 MAPK in CNS dysfunction and its potential as a therapeutic target. Specifically, little information is available on the quantitative contribution of microglial p381 MAPK signaling in vivo to up-regulation of proinflammatory molecules that lead to disease- relevant neuropathology, the importance of p381 MAPK in the beneficial reparative and remodeling responses of activated microglia, or the role of neuronal p381 MAPK in CNS dysfunction responses. We hypothesize that p381 (and not the closely related p382 MAPK isoform) is a key in vivo contributor to microglial inflammatory activation cascades that culminate in detrimental proinflammatory cytokine overproduction and subsequent neuronal/synaptic damage, and that suppression of p38 MAPK signaling in the microglia and/or neuron can lead to selective, beneficial outcomes. The field has been limited in its ability to pursue these questions because of the lack of CNS-penetrant, selective, small molecule p381 MAPK inhibitors. Our development of a novel, orally bioavailable, brain-penetrant, selective, small molecule p381 MAPK inhibitor (compound 069A) that attenuates hippocampal proinflammatory cytokine overproduction and leads to improved neurologic outcomes in a mouse CNS injury model now provides the opportunity to address these critical questions about p38 MAPK and CNS dysfunction in vivo as well as in vitro or in situ. We will use this unique chemical biology tool and novel knock-out (KO) and drug-resistant knock-in (KI) mice to pursue several important mechanistic investigations. First, we will test the importance of p381 MAPK and p382 MAPK in vivo through the use of microglial p381 conditional KO and p382 global KO mouse models subjected to stressor stimuli. The temporal onset and profile of microglial activation and synaptic dysfunction responses will be determined. Second, we will complement these in vivo studies by using cell culture models in order to explore in more detail the relative contributions of p38 MAPK isoforms in microglia and neurons. We will utilize microglial-neuronal co-cultures combined with pharmacological and genetic knock-down approaches to determine the importance of microglial and neuronal p38 MAPK isoforms to stressor-induced responses. Successful completion of this project will provide mechanistic insight into the role of the key regulatory protein, p381 MAPK, in microglial activation and neuronal damage caused by stressors that induce CNS pathophysiology, and the contribution of microglial vs. neuronal p381 MAPK to the CNS dysfunction responses. In addition, the studies will elucidate the potential role of the highly related p38 MAPK isoform, p382, in the microglial and neuronal responses. Longer term, the knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations.